Malignant transformation of the breast and other organs is associated with dramatic changes in the microenvironment surrounding neoplastic cells, including a reactive fibrotic stroma characterized by increased production of inflammatory cytokines, excessive accumulation of extracellular matrix (ECM), and an increase in tissue stiffness. Contractile myofibroblasts are key mediators of the biochemical and biophysical properties of the fibrotic tumor microenvironment. Additionally, the transdifferentiation of myofibroblasts from tissue cells and their subsequent activation is controlled by a combination of soluble factors and contractile tension. The increased tissue stiffness associated with fibrosis may thereby generate a positive feedback loop to facilitate tumor progression and metastatic invasion; delineating the microenvironmental effects and effectors will require sophisticated, tractable model systems. Here we describe the development of an experimental model that can define how alterations of biochemical and biophysical cellular microenvironment can stimulate myofibroblast development and activation, and how formation and activation of myofibroblasts in tissue structures affects progression to malignancy. In Specific Aim 1, we will determine the biochemical and biomechanical requirements of the substratum microenvironment for the transdifferentiation process. In Specific Aim 2, we will use a novel three- dimensional microlithography-based organotypic culture mimetic of the mammary epithelial ductal network to determine how myofibroblast transdifferentiation affects the microenvironment of the duct at the biochemical, mechanical, and cell population levels. Given that the presence of fibrotic foci in breast tumors correlates with metastasis and negative prognosis, and might hinder the efficacy of tumor therapies, the new physiologically relevant models developed in this work will have significant impact for discovery and evaluation of novel therapeutic targets to combat fibrosis genesis and tumor progression. PROJECT RELEVANCE: Repair of tissue damage involves the generation of specialized fibrous tissues that assist in tissue remodeling; deregulation or inappropriate activation of these repair processes can lead to fibrosis. Increasing evidence suggests that tissue fibrosis is a significant risk factor for development of cancer of the breast, lung, and many other organs. We present here a three-dimensional, microlithography-based model that can be used to break down the key steps involved in the earliest stages of fibrosis genesis. [unreadable] [unreadable] [unreadable]